Field of the Invention
The present technology relates to probes and methods of use thereof for monitoring aquatic environmental quality. More specifically, the technology relates to Mytilus edulis and Mytilus galloprovincialis probes and methods of use thereof to quantify stress levels in these invertebrates as a measure of anthropogenic or environmental variables in the water.
Description of the Related Art
Ocean coastlines around the world are under increasing pressure from urbanization, industry, recreation, aquaculture and climate change impacts. The presence of stressing agents from environmental, for example, but not limited to, temperature, salinity, food availability, or oxygen levels, biological, (for example, but not limited to bacteria or toxic algae) and anthropogenic sources; (for example, but not limited to wastewater, pollution, poor resource management or aquaculture practices) can affect the marine environment. These pressures require effective diagnostic tools to continually monitor coastal ecosystem health and function. Shellfish are a critical part of coastal and estuarine ecosystems and as sessile, filter-feeding animals, act as the “canary in the coalmine” of our coastal environment, providing a living warning of dangers to people on our coasts where human populations are concentrated. In North America, three such shellfish—mussels, oysters and Zebra mussels—have become sentinel or indicator species for monitoring environmental quality on the Pacific and Atlantic coasts from Alaska to Hawaii and from the Great Lakes to Puerto Rico. This “Mussel Watch” program has run continuously since 1986, includes 300 active monitoring sites and assesses 140 contaminants. Because shellfish are well distributed along coasts and are sessile, they are better integrators of contaminants in any given area. They are good indicators for environmental quality because their tissues respond to changes in ambient environmental levels, their wide geographical distribution, and their sessile nature make them excellent in situ bioaccumulators of pollution, providing valuable information on changes in coastal environments.
The global market for instrumentation to test and monitor wastewater is expected to reach $10 billion by 2016 as regulations in all jurisdictions become increasingly stringent and enforcement mechanisms improve. The Canadian economy relies heavily on fish and seafood—in 2004 these aquatic resources were the single largest export food commodity, by value, in Canada. Canada is the fifth largest seafood exporter in the world with more than $4.3 billion in value in 2005.
Uses of indicator species, as currently practised through well established programs like Mussel Watch, have become standard practice for monitoring the receiving environment. However, most of the analysis techniques rely on physical and chemical tests of the animal tissues, which are extremely slow and inefficient, and provide limited information on animal health and function.
In the US, biomonitoring is required under National Pollution Discharge Elimination System permits, which may include freshwater, marine or estuarine species. Facilities that process over a million gallons of wastewater per day are required to test water samples daily for toxicity and smaller facilities typically run these tests monthly. Effluent bioassays, biomonitoring and Whole Effluent Toxicity (WET) testing are all terms used to describe testing of wastewater discharge with aquatic organisms to assess the discharge's toxicity. WET tests are today's most common test for toxicity, but provide no information on the biological impacts of toxicity levels in ecosystems. In a WET test, organisms are exposed to various effluent concentrations for a specified time period in order to estimate toxicity. Sewage outflow water is used in the laboratory to simulate what happens in the natural environment. The most commonly used organisms for these tests are fathead minnows and an invertebrate—Ceriodapahnia dubia. Acute tests measure the concentration of test material that produces a lethal outcome during a 48-96 hour period. Chronic tests estimate the concentration of effluent that interferes with growth, development and reproduction over 4-7 days—the life cycle or life stage of the organism. WET tests average about $100/test in the US market.
When WET tests reveal the presence of a toxin and that toxin requires further identification, then the waste water facility must conduct a TIE and/or TRE study. A TRE—Toxicity Reduction Evaluation—is a systematic evaluation of the wastewater effluents to determine sources of toxicity and how to control this. This may include chemical screens, process reviews, evaluation of the facility's process performance and TIE—Toxicity Identification Evaluation. The objective of TIE is to characterize and identify the compound(s) causing toxicity. In TIE, effluent samples are manipulated to remove suspect chemicals and then re-test them to see if the toxicity remains, which provides clues to the analyst as to the source of the toxicity. TIE tests typically cost $1,000 each in the US market.
According to a Chief Plant Operator at a US waste water treatment plant, “it makes sense to offer a test similar to WET, but that is able to gather more nuanced information . . . at the depth offered by molecular analysis. End-users may find a new type of toxicity testing appealing if it were capable of gathering data on components beyond those for which testing is currently mandated. Other components of interest include: metals, chemical toxins, microbes, hormones or pharmaceuticals. A new method should be able to detect the presence of these components in trace amounts. End-users at water treatment plants may be interested in a micro-array if it can somehow identify toxins early, in a way that prevents drastic follow-up attempts to purify the water in the presence of toxins. If it could be demonstrated that this test can help prevent problems with toxins before reactionary methods become necessary, then end-users may be willing to purchase this type of technology before it is mandated by EPA.” A Lab Manager in the Ecology Division of a US environmental consulting firm, said that “one near-term application that may not require EPA approval is to use this new technology to reduce the number of TIE procedures that have to be performed once a toxin has been identified. If the new technology could reduce the number of TIE tests required to isolate toxins, then this would save companies time and money.”
At present tools for wastewater management using bivalves include visual indicators of acute and chronic mortality phases and other indicators such as shell growth, which have proven to be highly variable and therefore not always informative. To assess the effects of contaminants, histology assays have been developed that include the evaluation of lysosomal membrane stability and response indices. Biochemical assays include acetylcholinesterase activity, metallothionein content, vitellogenin, superoxidase dismutase, glutathione peroxidase levels and the accumulation of heat shock proteins. These assays are relatively difficult, time consuming to conduct, and each test provides information on only the end point of one gene which limits their usefulness in environmental monitoring. In addition, analysis of pollutant concentrations in sediments provides information on contamination levels, but not the effects on organism function, and benthic biodiversity analysis provides information of detrimental impacts only after community structure has shifted.
Microarrays have been used to measure gene expression in many systems. This is more sensitive than measuring biomarkers at the protein level. Analysis of gene expression profiles is increasingly used to evaluate the biological effects of stress on aquatic animals. Using Reverse Transcription real-time quantitative Polymerase Chain Reaction (qRT-PCR) in mussel studies, only limited numbers of genes have been examined, for example five genes from M. galloprovincialis in Li et al., 2010 (“Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection.” Fish & Shellfish Immunology 28(4): 640-648), five in Veldhoen et al., 2011 (“Relationship between mRNA biomarker candidates and location near a marine municipal wastewater outfall in the benthic indicator species Modiolus modiolus (L.).” Aquatic Toxicology 105(1-2): 119-126) and twelve in Veldhoen et al., 2009 (“Gene expression profiling in the deep water horse mussel Modiolus modiolus (L.) located near a marine municipal wastewater outfall.” Aquatic Toxicology 93(2-3): 116-124) from Modiolus modiolus. 
There is a need to improve environmental monitoring in coastal waters. Testing methods should be rapid, accurate and inexpensive. It would be advantageous to have a suite of biomarkers to facilitate a comprehensive understanding of organism health as it relates to the health of marine environments. These could then be employed to monitor the marine environment. It would also be advantageous if the biomarkers could support versioning to keep current and meet commercial market requirements, have broad global species coverage and have the capability to monitor a broad range of environmental stressors.